1. Field of the Invention
The present invention relates generally to immunointeractive molecules and more particularly antibodies which bind to vascular endothelial growth factor-B (VEGF-B) or its functional or structural equivalent and inhibit the biological activity of VEGF-B. In particular, the present invention relates to deimmunized molecules such as humanized or human antibodies that bind to VEGF-B and inhibit the biological activity of VEGF-B. These antibodies have uses in the treatment or prevention of diseases associated with perturbations in normal vasculogenesis or angiogenesis or vascular remodelling. The present invention further contemplates a method of modulating diseases associated with perturbations in normal vasculogenesis or angiogenesis or vascular remodelling by the administration of the subject antibodies. The present invention further provides an assay system useful for identifying antibodies which bind to VEGF-B and block the biological activity of VEGF-B. Accordingly, a method of screening for inhibitors of the biological activity of VEGF-B is also provided.
2. Description of the Prior Art
The reference to any prior art in this specification is not and should not be taken as an acknowledgment or any form of suggestion that the prior art forms part of the common general knowledge in any country.
Bibliographic details of the publications referred to in this specification are also collected at the end of the description.
The normal growth of new blood vessels, or physiological angiogenesis, is an essential step in vertebrate growth and development as well as in the repair of wounds and bone fractures. This process of blood vessel formation and remodelling is kept in close control by pro- and anti-angiogenic molecules, but perturbations in the process can occur. Abnormal or pathological angiogenesis occurs when the balance of blood vessel growth is disturbed and is a contributory factor in the development of a wide range of diseases, such as rheumatoid arthritis (Kasama et al., Arthritis Rheum. 44(11): 2512-2524, 2001) and malignant angiogenic tumours and cancer-cell metastases (Liu et al., J. Surg. Res. 102(1): 31-34, 2002).
Growth and remodelling of the vascular system are mediated by a diverse collection of polypeptide growth factors. One such group is the peptide family known as vascular endothelial growth factors (VEGFs) (Tuder et al., J. Pathol. 195(3): 367-374, 2001). The VEGFs constitute a group of structurally and functionally related growth factors that modulate many important physiological functions of endothelial cells. The mammalian members of the VEGF family identified to date include VEGF-A, VEGF-B, VEGF-C, VEGF-D and placental growth factor.
The various homologues of VEGF differ slightly in the roles they play during the various developmental stages and also in response to vascular trauma. This is indicated by the variations in temporal and spatial release of the various VEGFs during physiological events such as embryonic development, regulation of capillary growth in normal and pathological conditions in adults, and in the maintenance of the normal vasculature. For example, VEGF-A is a potent mitogen that plays a vital role in vasculogenesis and angiogenesis during development (Brown et al., Am. J. Physiol. Lung Cell Mol. Physiol. 281(4): L1001-1010, 2001). It is also vital for revascularization during repair of dermal wounds (Mat this et al., Am J. Pathol 160(1): 289-296, 2002) and regrowth of vasculature following bone fractures (Street et al., J. Orthop. Res. 19(6): 1057-1066, 2001).
Gene knockout experiments have found that VEGF-B is not essential for the growth and development of the peripheral vascular system, although it is involved in the normal development of the coronary vasculature (Bellomo et al., Circ. Res. 86(2): E29-35, 2000). It also plays a part in physiological responses to ischemia and vascular occlusion (Bellomo et al. [2000; supra]). VEGF-B is also implicated in a number of pathological angiogenic conditions such as pulmonary hypertension (Rich et al., J. Heart Lung Transplant 21(1): 159, 2002), the growth of angiogenic tumors (Li et al., Growth Factors 19(1): 49-59, 2001) and the spread or metastases of cancer cells, possibly through its effects on plasminogen activation (Gunningham et al., J. Pathol. 193(3): 325-332, 2001).
The actions of VEGF-B are mediated through the receptor tyrosine kinase VEGF receptor-1 (VEGF-R1). VEGF-R1 is also referred to as Flt-1 and its extracellular domain is characterized by seven immunoglobulin-like regions (Ma et al., Biotechnol. Appl. Biochem. 34 (Pt 3): 199-204, 2001), referred to as Ig domains 1-7.
The suspected role of VEGF-B in pathological angiogenesis has made this growth factor a desirable control point in the treatment of a number of diseases. Biological profiling of VEGF-B has, however, been limited by a lack of simple in vitro assay systems.
When further characterizing the biological effects of VEGF-B, the inventors faced difficulties with sub-optimal cell-based assays. Reports of activity of VEGF-B on endothelial cells, including stimulation of proliferation and induction of mRNA for uPA and PAI-1 have subsequently been attributed to contaminating heterodimer and lipopolysaccharide, respectively. The present inventors have now devised a novel, cellular based assay for VEGF-B activity which is based on the development of a chimeric fusion molecule encoding the extracellular portion of the VEGF-B receptor. The assay is also useful for identifying modulators of VEGF-B-Flt-1-mediated signalling.
Antibodies to VEGF-B may potentially act as antagonists of VEGF-B biological activity. In accordance with the present invention, antibodies are identified which bind to VEGF-B and block VEGF-B binding to VEGF-R1, thereby inhibiting the biological activity of VEGF-B.